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* This work was supported by Spanish Government Grants FIS 2000/230, FIS 2002/PI20265, and CAM 8.5/49/2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Recipient of a predoctoral fellowship (Becas de Formación en Investigación). § Recipient of a postdoctoral fellowship (Comunidad Autonoma de Madrid).
Glutathione (GSH) depletion is the earliest biochemical alteration shown to date in brains of Parkinson's disease patients. However, data from animal models show that GSH depletion by itself is not sufficient to induce nigral degeneration. We have previously shown that non-toxic inhibition of GSH synthesis with l-buthionine-(S,R)-sulfoximine in primary midbrain cultures transforms a nitric oxide (NO) neurotrophic effect, selective for dopamine neurons, into a toxic effect with participation of guanylate cyclase (GC) and cGMP-dependent protein kinase (PKG) (Canals, S., Casarejos, M. J., de Bernardo, S., Rodríguez-Martín, E., and Mena, M. A. (2001) J. Neurochem. 79, 1183–1195). Here we demonstrate that arachidonic acid (AA) metabolism through the 12-lipoxygenase (12-LOX) pathway is also central for this GSH-NO interaction. LOX inhibitors (nordihydroguaiaretic acid and baicalein), but not cyclooxygenase (indomethacin) or epoxygenase (clotrimazole) ones, prevent cell death in the culture, even when added 10 h after NO treatment. Furthermore, the addition of AA to GSH-depleted cultures precipitates a cell death process that is indistinguishable from that initiated by NO in its morphology, time course, and 12-LOX, GC, and PKG dependence. The first AA metabolite through the 12-LOX enzyme, 12-hydroperoxyeicosatetraenoic acid, induces cell death in the culture, and its toxicity is greatly enhanced by GSH depletion. In addition we show that if GSH synthesis inhibition persists for up to 4 days without any additional treatment, it will induce a cell death process that also depends on 12-LOX, GC, and PKG activation. In this study, therefore, we show that the signaling pathway AA/12-LOX/12-HPETE/GC/PKG may be important in several pathologies in which GSH decrease has been documented, such as Parkinson's disease. The potentiating effect of NO over such a signaling pathway may be of relevance as part of the cascade of events leading to and sustaining nerve cell death.
In a number of neurological disorders, including Parkinson's disease (PD),
) have been found in PD brains. Whether such alterations are a primary cause of the disease, part of the degenerative mechanism, or secondary to the cell death process remains to be resolved.
In cell cultures and animal models of several diseases, including PD, these alterations may contribute to neuronal degeneration. Furthermore, synergistic interactions between them have been demonstrated and greatly enhance the neurodegenerative process (
GSH depletion is the earliest biochemical alteration shown to date in PD brains. It seems to appear before neurodegeneration in incidental Lewy bodies disease, which is considered to be the presymptomatic manifestation of PD (
). However, the reduction of GSH levels may rather enhance the susceptibility of DA cells to the toxicity of other insults, promoting the neurodegenerative process. For example, GSH depletion induced by l-buthionine-(S,R)-sulfoximine (BSO) treatment, a selective GSH synthesis inhibitor (
We have described previously a culture model in which one of these interactions was clearly stated. In midbrain cultures, the short lived NO donor diethyl-amine/nitric oxide-complexed sodium (DEA/NO), at doses of 25 and 50 μm, selectively increases the number of tyrosine hydroxylase positive (TH+) cells, TH+ neurite processes, DA syntheses, and [3H]DA uptake (
). In addition, under GSH-decreased conditions, neurotrophic doses of NO trigger a programmed cell death with dependence on guanylate cyclase (GC) and cyclic GMP-dependent protein kinase (PKG) activation. Also, we have shown that the GC activation that mediates cell death is not produced by NO (
In the present work we go far inside the mechanism related to the neurotoxic interaction between decreased GSH levels and NO by exploring the possible participation of arachidonic acid (AA) metabolism. AA is an important component of membrane lipids that can activate several signaling pathways directly by itself or by its metabolites through lipoxygenase (LOX), cyclooxygenase (COX), or epoxygenase pathways (
). Here we show that NO and GSH synergically interact at the AA metabolic pathway to induce cell death in midbrain cultures.
Culture Media—Dulbecco's modified Eagle's medium with high glucose (4.5g/liter), Ham's F12 nutrient mixture, Eagle's minimal essential medium with Earl's salts, and Leibovitz's l-15 medium, all of which were supplemented with l-glutamine, fetal calf serum (FCS), sodium pyruvate, and l-glutamine, were purchased from Invitrogen. Glucose (45%), insulin, putrescine, progesterone, and sodium selenite were from Sigma, and human transferrin (30% iron-saturated) was from Roche Applied Science.
Antibodies—Rabbit polyclonal anti-mouse leukocyte 12-lipoxygenase antibody was from Alexis (Carlsbad, CA); mouse monoclonal anti-tyrosine hydroxylase (TH) antibody was from Chemicon (Temecula, CA); O1 was obtained from hybridoma supernatants (
); polyclonal anti-GFAP antibody, raised in rabbits, was from DAKO (Glostrup, Denmark); the anti-microtubule-associated protein 2a + 2b (MAP-2) antibody, the mouse monoclonal anti-β-actin antibody, and anti-rabbit IgG conjugated with tetramethylrhodamine isothiocyanate (TRITC) were purchased from Sigma; anti-mouse Ig fluorescein was from Jackson ImmunoResearch Laboratories (West Grove, PA); and anti-mouse IgM Alexa Fluor® 488 was from Molecular Probes (Eugene, OR).
Chemicals—Poly-d-lysine, p-phenylenediamine, bis-benzimide, BSO, dimethyl sulfoxide (Me2SO), DTNB, and reduced and oxidized forms of glutathione and AA were from Sigma. DEA/NO was from Alexis. NADPH, the cytotoxicity detection kit (lactate dehydrogenase, or LDH), cell proliferation kit I (3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide, or MTT), and GSH reductase (GR) were from Roche Applied Science. 12-HPETE, 12-HETE, and LY-83583 were from Biomol (Plymouth, PA). KT5823, baicalein, nordihydroguaiaretic acid (NDGA), indomethacin, and clotrimazole were from Calbiochem. The BCA protein assay kit was from Pierce. All other reagents were of the highest purity commercially available from Merck or Sigma.
Neuronal Culture—Neuronal enriched cultures from embryonic Sprague-Dawley rat midbrain E-14 (crown rump, length 10–12 mm) were obtained and prepared as described previously (
). The cells were seeded in Dulbecco's modified Eagle's medium with 15% fetal calf serum at a density of 105 cells/cm2 in multiwells or glass cover slides previously coated with poly-d-lysine, 4.5 μg/cm2, in 0.1 m borate buffer, pH 8.4. The cultures were kept in a humidified chamber at 37 °C in a 5% CO2 atmosphere. Twenty-four hours after plating, the cells were changed to a serum-free defined medium (EF12) as reported elsewhere (
). EF12 consisted of a 1:1 (v/v) Eagle's minimal essential medium and nutrient mixture of Ham's F-12 supplemented with d-glucose (6 mg/ml), insulin (25 μg/ml), transferrin (100 μg/ml), putrescine (60 μm), progesterone (20 nm), and sodium selenite (30 nm).
Experimental Treatments—In the experiments designed to study interactions between NO and GSH depletion on cell viability, the cells, after 4 days in culture, received 20 μm BSO or vehicle, and then, on the 5th day, pre-established groups were treated with the NO donor DEA/NO (50 or 100 μm) for an additional 24 h. Enzyme inhibitors for the three pathways of the AA metabolism, i.e. the LOX pathway (NDGA and baicalein), the COX pathway (indomethacin), and the epoxygenase pathway (clotrimazole and proadifen) or its corresponding solvents, were routinely added 30 min before DEA/NO treatment or up to 10 h later. For experiments with AA and 12-LOX metabolites, cultures were treated with BSO as above and, on the 5th day, received several doses of AA, 12-HPETE, 12-HETE, or solvent for an additional 24 h. Finally, experiments for time course effects of 100 μm DEA/NO or 20 μm BSO on 12-LOX expression, alone or in combination, were also depicted.
The long term effect of GSH synthesis inhibition on cell viability was also studied. The cells received 20 μm BSO as above, and the treatment proceeded for up to 4 days (the 8th day in vitro). In this experimental design, enzyme inhibitors or vehicles were added to the culture on the 5th day in vitro. The participation of 12-LOX in neurotoxicity induced by higher doses of NO under normal GSH homeostasis was investigated by pretreating cultures with NDGA or baicalein 30 min before the addition of 200 and 400 μm DEA/NO.
Immunocytochemistry—DA neurons were characterized by immunostaining with a mouse monoclonal anti-TH antibody (1:100), astrocytes with a rabbit polyclonal anti-GFAP antibody (1:500), and oligodendrocytes with monoclonal anti-O1 (1:10) (
). To detect all neurons in the culture, a mouse monoclonal anti-MAP-2 antibody (1:250) was used. For TH, GFAP, and MAP-2 immunostaining, cultures were fixed with 4% paraformaldehyde, washed in 0.1 m phosphate-buffered saline (PBS), pH 7.4, permeabilized with ethanol-acetic acid (19:1), and incubated at 4 °C for 24 h with primary antibodies diluted in PBS containing 10% fetal calf serum. Fluorescein- and rhodamine-conjugated secondary antibodies were employed to visualize positive cells under fluorescent microscopy. For oligodendrocyte detection, anti-O1 antibody was added directly (1:10) to living cells and incubated for 15 min at room temperature, washed in PBS, and fixed with 4% paraformaldehyde previous to anti-mouse IgM Alexa Fluor® 488 development. The number of immunoreactive cells was counted in one-seventh of the total area of the cover slides. The cells were counted in predefined parallel strips using a counting reticule inserted in the ocular.
Cell Viability Measurements—Mitochondrial activity was measured with the MTT assay. Cells were grown on 24-well culture plates with 500 μl of defined medium and treated with various reagents according to the experimental design. The MTT assay measures the ability of cells to metabolize MTT. At the end of the treatment period, 300 μl of culture medium were removed from each well, and 20 μl of MTT solution (5 mg/ml) were added and incubated for 1 h. At this time, 200 μl of solubilization solution (10% SDS in HCl, 0.01 m) were then added to the wells, and, after 24 h of incubation at 37 °C, 100 μl were transferred onto 96-well microtiter plates, and the absorption value at 540 nm was measured in an automatic microtiter reader (Spectra Fluor, Tecan).
Chromatin condensation was assessed by DNA staining with bisbenzimide (Hoechst 33342). Cells growing on cover slides were fixed in 4% paraformaldehyde, and the nuclei were stained with bis-benzimide added in the anti-fading solution (3 × 10–6m, final concentration) (
) and expressed as a percentage versus detergent-extracted controls (100% cytotoxicity). In our system, LDH release to the culture medium correlates with cell death measured by a trypan blue dye exclusion assay (
Western Blot Analysis—Primary midbrain cultures were homogenized with a sonicator in buffer containing 20 mm Tris HCl, 10 mm acetyl lysine, 1 mm dithiothreitol, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 mm benzamidine, leupeptin, aprotinin, and pepstatin (5 μg/ml each), and 0.25% Nonidet P-40, pH 7.4, and then centrifuged at 12,000 × g for 30 min at 4 °C. The supernatant was used for protein determination by the BCA protein assay kit and for electrophoretical separation. Samples (30 μg) were added to SDS sample loading buffer, electrophoresed in 10% SDS-polyacrylamide gels, and then electroblotted to 0.45-μm nitrocellulose membranes. For immunolabeling, the blots were blocked with TTBS (20 mm Tris-HCl, pH 7.6, 137 mm NaCl plus 0.1% (v/v) Tween 20, and 5% dry skimmed milk) for 1 h at room temperature. After blocking nonspecific binding, the membranes were incubated with rabbit anti-12-LOX (1:2000) and mouse anti-β-actin (1:10000) in blocking solution overnight at 4 °C. The blots were developed by chemiluminiscence detection using a commercial kit (Amersham Biosciences) and quantified by computer-assisted video densitometry. β-actin was employed as a control of charge.
Glutathione Measurements—Total glutathione levels were measured by the method of Tietze in 1969 (
). Briefly, 1 × 105 cells were washed with PBS, lysed in 100 μl of 3% perchloric acid (PCA) for 30 min at 4 °C, centrifuged, and the supernatants were neutralized with 4 volumes of 0.1 m NaH2PO4 and 5 mm EDTA, pH 7.5. Fifty microliters of the resulting supernatants were mixed with DTNB (0.6 mm), NADPH (0.2 mm), and glutathione reductase (1 unit), and the reaction was monitored in a P96 automatic microtiter reader at 412 nm for 6 min. Oxidized glutathione (GSSG) was measured in the cells by the method of Griffith in 1980 (
). Briefly, after perchloric acid extraction and pH neutralization, GSH was derivatized with 2-vinylpyridine at room temperature for 1 h, and the reaction was carried out as described above. GSH was obtained by subtracting GSSG levels from total glutathione levels.
Statistical Analysis—The results were statistically evaluated for significance with one-way analysis of variance (ANOVA) followed by the Newman-Keuls multiple comparison test as a post hoc evaluation. Differences were considered statistically significant when p < 0.05.
Inhibitors of Lipoxygenase Block NO-triggered Cell Death in GSH-depleted Cultures—Midbrain cultures treated for 24 h with 20 μm BSO decreased its GSH content from 17.5 ± 0.4 μg/mg of protein to 3.7 ± 0.1 μg/mg of protein (≈20% versus control). In these GSH-depleted conditions, treatment of cultures for an additional 24 h with the NO donor DEA/NO at doses previously shown to be neurotrophic for DA neurons (50 μm) results in extensive cell death in the culture (Fig. 1, a and b). The death process is characterized by a 75–85% loss of mitochondrial activity measured by MTT assay, a 30–35% increase in LDH release to the culture medium, and 70–80% of cells with chromatin condensation without DNA fragmentation (Figs. 1, a and b, and 2; and data not shown), as shown previously (
). However, this rate of GSH synthesis inhibition by itself did not induce cell death in the culture at 48 h (Fig. 2, a and b) or at 72 h (data not shown) in accordance with previously reported data for neuronal enriched midbrain cultures (
). To investigate the possible role of lipoxygenase in cell death in the above conditions, we first used the general LOX inhibitor NDGA. Added 30 min before the NO treatment, NDGA preserves cell viability in the culture (75–80% of controls viability) and totally blocks LDH release and chromatin condensation induced by BSO + DEA/NO treatment (Figs. 1c and 2, a–c). The concentration required for maximal protection was 0.5 μm, being toxic to the culture up to 2.5 μm (data not shown). Because 12-LOX is the predominant brain isoform of this enzyme (
), we tested baicalein, a selective 12-LOX inhibitor, for cell viability assays. As seen for NDGA, baicalein rescues all viability parameters measured in the culture with maximal protection observed at 2.5 μm (Figs. 1d and 2, a–d). Both inhibitors protect against cell death induced by 50 μm as well as 100 μm DEA/NO in BSO-pretreated cultures (Fig. 2, a–d) without interfering with BSO-induced GSH depletion (data not shown).
In contrast, when indomethacin, a specific inhibitor of cyclooxygenases with an IC50 of 1 μm, was used at concentrations up to 50 μm, no protection from cell death was seen (Figs. 1e and 2, a and b). In addition, indomethacin, which is not toxic for fetal midbrain cultures at the doses used here, increases the toxicity of NO in GSH-depleted midbrain cultures (Fig. 2, a and b). Two inhibitors of epoxygenase, clotrimazole and proadifen, were also proven for cell death protection. Clotrimazole, with an IC50 of 0.4 μm, used at concentrations up to 5 μm, did not show any protective effect (Figs. 1f and 2, a and b). In the same way as indomethacin, non-toxic doses of clotrimazole further increased cell death in the culture (Fig. 2, a and b). However, the other epoxygenase inhibitor, proadifen, provides partial neuroprotection at 10 μm (Fig. 2, a and b). Although widely used as an epoxygenase inhibitor, proadifen also inhibits LOX activity (
). In view of the above results, the proadifen protective effect is probably due to 12-LOX inhibition.
We have shown previously that NO-induced cell death on GSH-down-regulated midbrain cultures required GC activation. The protection afforded by GC inhibitors occurred up to 10 h after the addition of NO (
). To determine the temporal correlation between GC inhibition and LOX inhibition on cell death prevention, we tested the efficacy of baicalein to protect cells when it is added to the culture 2, 6, and 10 h after NO treatment. As shown in Figs. 2, d and e, the 12-LOX inhibitor efficiently prevents cell death at any time it is used, similar to what occurs with GC inhibitors. The same results were obtained with the LOX inhibitor NDGA (data not shown).
Some antioxidant properties have been described for baicalein and NDGA that are independent of the LOX inhibition (
). Because the protective effect of LOX inhibitors persists up to 10 h after NO treatment, but ascorbic acid protection is lost after 2 h (Fig. 2e), an antioxidant role of LOX inhibitors on cell death prevention is ruled out in our model.
Effect of BSO and DEA/NO Treatment on 12-LOX Protein Levels—Li et al. (
) have shown previously that GSH depletion, induced by glutamate treatment in primary immature cortical cultures, induces a 2- to 3-fold increase in 12-LOX protein. To test this possibility in midbrain cultures depleted of GSH, we performed Western blot analyses of the 12-LOX protein. As shown in Fig. 3, GSH depletion for up to 48 h, alone or in combination with NO treatment, did not vary 12-LOX protein levels. Furthermore, NO alone (100 μm DEA/NO) did not change 12-LOX levels after either 1, 8, or 16 h of treatment (Fig 3b). We used β-actin as a control of charge, and no statistical differences were found in 12-LOX/β-actin ratios between any experimental group.
12-LOX Inhibition Protects All Cell Types in the Culture— Immunocytochemical characterization of cell death induced by BSO + DEA/NO treatment in fetal midbrain cultures shows that the most affected cell types were neurons (MAP-2+ cells) and oligodendrocytes (O1+ cells) (Fig. 4). Among neurons, TH+ cells, although very affected, were more preserved (Figs. 4 and 5), in accordance with previously reported data (
). Astrocytes (GFAP+ cells) were the most resistant cell type in the culture. When the ability of LOX inhibitors (NDGA and baicalein) to protect against cell death was studied for different cell populations, we observed that all cell types in the culture were protected from toxicity (Figs. 4 and 5).
Arachidonic Acid and 12-HPETE, but Not 12-HETE, Reproduce in GSH-depleted Midbrain Cultures, All Features of Cell Death Triggered by NO—Incubation of midbrain cultures for 24 h with increasing concentrations of AA resulted in no sign of toxicity in doses ranging from 0.3 to 6 μm, and the cultures were very slightly affected at a dose of 12 μm. By contrast, after GSH depletion, AA induced cell death in the culture in a dose-dependent manner, reaching 100% toxicity from 3 μm (Fig. 6). Cell death is characterized by shrinkage, rounded cells with chromatin condensed peripherally in the nucleus without DNA fragmentation (Fig. 6c, and data not shown), loss of mitochondrial activity (measured by MTT assay; Fig. 6a), and the breakdown of plasmatic membrane (measured as LDH released to culture medium; Fig. 6b); this is similar to what occurs with NO in GSH-depleted cultures (Fig. 1 and 2). Also, the temporal course of the cell death process is similar to that initiated by NO, because there was no change in cell viability for at least 16 h after AA treatment (data not shown). Furthermore, LOX inhibitors (NDGA and baicalein), but not cyclooxygenase (indomethacin) or epoxygenase (clotrimazole) inhibitors, protect against AA-induced cell death (Fig. 6d). Finally, cell death was also blocked by the GC inhibitor LY83583 and the PKG inhibitor KT5823 (Fig. 6d) in the same way that NO triggered cell death.
These results demonstrate that GSH depletion by itself potentiates AA metabolism through the 12-LOX pathway and that AA limits LOX activity in our model, because AA supplementation is sufficient to induce cell death in the culture. In addition, the results indicate that the cell death process initiated by AA under GSH-decreased conditions is indistinguishable from that induced by NO and suggest that a relationship may exist between these two events.
Next we tested the ability of 12-HPETE, the first AA metabolite through the 12-LOX pathway, to induce cell death in solvent or BSO-pretreated cultures. Incubation of control cultures with increasing concentrations of 12-HPETE resulted in a loss of viability from 3 μm, and BSO pretreatment greatly increased its toxicity (Fig. 7, a and b). Unlike NO or AA in GSH-decreased cultures, 12-HPETE did not require long exposure times for toxicity, and cell death was visible by phase-contrast microscopy (data not shown) as soon as 2 h after treatment. This observation suggests that 12-HPETE participates in the execution phase of the cell death process. In addition, 12-HETE, the main downstream product of 12-HPETE in brain, was unable to induce cell death in control or GSH-depleted cultures (Fig. 7b). Overall, these data indicate that 12-HPETE is the metabolite responsible for cell death induction by NO and AA in our model.
GSH Depletion Rather than NO Determines the LOX/GC/PKG Dependence of Cell Death Induced by BSO + DEA/NO Treatment—GSH depletion induced by 20 μm BSO results in no sign of toxicity in midbrain cultures after 48 h of treatment (Fig. 2). If BSO treatment persists, loss of viability occurs on the 4th day without NO addition (Fig. 8). Cell death produced in these experimental conditions is prevented by 12-LOX, GC, and PKG inhibition (Fig. 8). The toxicity of NO for DA neurons in midbrain cultures without pharmacological alteration of the GSH system is observed from 200 to 400 μm DEA/NO, but this toxic effect is not dependent on GC and LOX activation (Ref.
, and data not shown). Overall, these data suggest that GSH depletion did not favor NO to be toxic due to the loss of one of the most important antioxidant systems in the cell, but rather that NO precipitates the cell death process that takes place in GSH-decreased conditions and requires LOX, GC, and PKG activation.
The effect of GSH depletion on neuronal viability has been widely studied in several experimental models of neurodegenerative diseases. PD models have received special attention since a severe GSH depletion in the substantia nigra of PD patients has been described (
). In cell culture models, different tolerances to GSH depletion have been reported depending on cell type and the brain region used for primary cultures. Cell death in primary immature cortical neurons and the HT22 hippocampal nerve cell line is dose dependently induced by 50–95% GSH depletion after 12 h of BSO incubation, and the death process depends on 12-LOX and GC activation without PKG participation (
). As we show in Fig. 8, if GSH synthesis inhibition in neuronal enriched midbrain cultures persists up to 4 days (by 20 μm BSO treatment), it causes the death of the culture. Cell death in those GSH-depleted conditions is prevented by LOX, GC, and PKG inhibitors. Despite the differences in susceptibility, cell death induced by GSH depletion seems to be tightly linked with 12-LOX and GC activation in different culture models.
We have shown previously that the tolerance to GSH depletion of neuronal enriched midbrain cultures and DA neurons is lost after non-toxic NO addition. Neurotrophic doses of NO for DA neurons, when added after 24 h of 20 μm BSO pretreatment, precipitate cell death in a GC- and PKG-dependent manner (
). Here we demonstrate that 12-LOX is also central for this NO effect, because NDGA and baicalein prevent cell death in the culture. Other pathways of AA metabolism in our model are excluded, because cyclooxygenase and epoxygenase inhibitors did not confer any protection. Furthermore, they increase both NO- and AA-induced cell death. This potentiating effect may be related to a rise in AA availability for 12-LOX metabolism, as has been described for other systems (
). We conclude that GSH decrease and NO, interacting on AA metabolism through 12-LOX, cooperate to induce cell death in neuronal enriched midbrain cultures. Interestingly, midbrain cultures containing serum with a great proportion of glial cells become more sensitive to GSH depletion than neuronal enriched cultures and die after 48 h of BSO treatment (
). In agreement with the role of NO in potentiating BSO toxicity through the 12-LOX pathway, cell death in those glia-containing cultures is blocked by the NO synthase inhibitor l-nitro arginine methyl ester (l-NAME) and the LOX inhibitor NDGA (
The 12-LOX enzyme is clearly detected in primary midbrain cultures, but its synthesis is not regulated by the isolated or combined depletion of GSH and NO treatment. Nevertheless, the addition of AA to BSO-pretreated cultures precipitates neuronal cell death at doses in which AA is not toxic for midbrain cultures, and the effect is prevented by NDGA and baicalein, indicating that GSH depletion by itself is sufficient to activate 12-LOX. These observations are in agreement with previously reported data showing that GSH directly inhibits LOX (
The exact mechanism by which NO potentiates BSO toxicity in our neuronal enriched midbrain cultures needs further investigation. Cell death produced by AA supplementation in GSH-depleted cultures is indistinguishable from that induced by NO in its time course, morphology, and molecular signaling pathway (12-LOX/GC/PKG), supporting the notion that increased AA metabolism is behind NO effects and linking the 12-LOX metabolites of AA with GC/PKG activation and cell death. The fact that AA precipitates cell death in BSO-pre-treated cultures indicates that AA limits LOX activity; therefore, one possibility for NO action is to increase non-esterified AA availability for the 12-LOX metabolism. Several groups have implicated PLA2 activation and the decreased rate of AA re-esterification to the membrane as mechanisms for a NO-induced increase in free AA (
) and thus decreases ATP levels, this mechanism of free AA up-regulation is possibly operating in our model.
Several sources of evidence support the idea that 12-HPETE is the 12-LOX metabolite implicated in the cell death process. 12-HPETE induces cell death in midbrain cultures, and its toxicity is greatly enhanced by GSH depletion. It has been shown that 12-HPETE activates GC (
), and a NO-independent GC activation is needed for cell death induced by DEA/NO in GSH-decreased midbrain cultures. The toxicity of AA under GSH-depleted conditions as well as the toxicity of long term GSH synthesis inhibition also require GC activation. There is a good temporal relationship between GC inhibition and LOX inhibition on cell death prevention in our model. In addition, 12-HPETE plays a role in the execution phase of the cell death process, because its toxic effect is observed as soon as 2 h after treatment. Finally, 12-HETE, the main downstream metabolite of 12-HPETE without GC activation capacity, did not induce cell death in control or BSO-treated cultures. Although the reduction of 12-HPETE to 12-HETE is very rapid, and it has long been known to be a major metabolite of AA in brain (
), the 12-HPETE metabolism is not limited to its reduction and, therefore, the participation of other derived compounds such as hepoxilins in cell death may not be formally discarded.
Overall, the results with AA and 12-HPETE suggest that GSH decrease fulfills at least two functions related to lipid peroxidation in our model as follows: (i) increased 12-LOX activity; and (ii) increased 12-HPETE half-life by decreasing its reduction rate. The last hypothesis is based on the fact that GSH depletion potentiates 12-HPETE toxicity and on data showing that the GSH system participates in the reduction of lipid hydroperoxides into hydroxy acids (
). But another important function of GSH may be directly related to NO. GSH can compete with cellular targets of NO by conjugating with it to form nitrosoglutathione or by regenerating nitrosyl groups and, thus, limiting NO actions (
). All these data indicate that NO and AA are implicated in the degeneration of a nigro striatal system in rodents. Because GSH depletion potentiates MPTP toxicity in vivo, we propose that interactions between GSH, NO, and AA metabolisms may be important for DA cell toxicity in PD experimental models. In PD patients, along with GSH depletion, a marked increase of glial cells expressing inducible NO synthase in the substantia nigra has been described (
). This indicates the possibility that, in PD nigra, NO concentration increases in the vicinity of DA neurons in a general environment of GSH depletion. In combination, NO overproduction and GSH depletion may interact, reaching a toxic threshold responsible for DA degeneration. Our experiments suggest that, in such a scenario, intervention of the AA metabolism through the 12-LOX pathway may be beneficial to individuals suffering from PD. The model predicts that, under GSH-decreased conditions such as PD, any stimuli that increase non-esterified AA availability will also contribute to cell death through the 12-LOX pathway. In this way, pro-inflammatory cytokines (interleukin-1, tumor necrosis factor-α, and interferon-γ), which are increased in PD brains (